The overall objective of the project is an understanding of the mechanisms of mutation in human cells: what factors determine the site at which particular mutagens react; how does the cell remove damaged portions of the DNA and what are the limits to this removal; how does replication occur past lesions in the DNA and what are the biological consequences of such replication? We are currently engaged in two lines of work to attain this objective: 1) Use of an in vitro system of DNA synthesis using templates of phi X174 reacted with mutagens in order to determine the nucleotide sites at which synthesis is inhibited; and 2) studies with human lymphoblastoid cells on the mechanism of removal of O6-methylguanine adducts from DNA. In the coming year we plan three lines of work: a) Use of extracts from prokaryotic cells induced for in vivo bypass and use of single stranded binding proteins to attempt to convert our in vitro system into one which can bypass chemical lesions in the template DNA; b) development of methods for making large numbers of stable apurinic sites in phi X174 DNA in order to study the effect of particular apurinic sites on DNA synthesis in our in vitro system; and c) study of the substances involved in the removal of O6-methylguanine from the DNA of lymphoblastoid cells. Our current work indicates that some constituent, present in a class of lymphoid cells, is "used up" in the removal of this adduct much as has been reported in bacteria. We wish to develop methods to assay this constituent and perhaps to isolate it and characterize the removal reaction as it occurs in human cells.